Application of Gold Nanoparticles as Radiosensitizer for Metastatic Prostate Cancer Cell Lines

More than 50% of all prostate cancer (PCa) patients are treated by radiotherapy (RT). Radioresistance and cancer recurrence are two consequences of the therapy and are related to dose heterogeneity and non-selectivity between normal and tumoral cells. Gold nanoparticles (AuNPs) could be used as potential radiosensitizers to overcome these therapeutic limitations of RT. This study assessed the biological interaction of different morphologies of AuNPs with ionizing radiation (IR) in PCa cells. To achieve that aim, three different amine-pegylated AuNPs were synthesized with distinct sizes and shapes (spherical, AuNPsp-PEG, star, AuNPst-PEG, and rods, AuNPr-PEG) and viability, injury and colony assays were used to analyze their biological effect on PCa cells (PC3, DU145, and LNCaP) when submitted to the accumulative fraction of RT. The combinatory effect of AuNPs with IR decreased cell viability and increased apoptosis compared to cells treated only with IR or untreated cells. Additionally, our results showed an increase in the sensitization enhancement ratio by cells treated with AuNPs and IR, and this effect is cell line dependent. Our findings support that the design of AuNPs modulated their cellular behavior and suggested that AuNPs could improve the RT efficacy in PCa cells.


Introduction
Radiotherapy (RT) is one of the most used therapeutic approaches to treat PCa, which is one of the most commonly diagnosed non-skin cancers affecting men, and in fourth place for cancer mortality in males [1]. Approximately 50% of all patients receive RT at some point during treatment [2]. RT is a therapeutic approach that uses ionizing radiation (IR) to induce cell damage and kill cancer cells. The main goal of this therapy is to deliver a Additionally, the morphology of the nanoparticles was assessed by transmission electron microscopy-TEM ( Figure 1G-I). AuNPs were evaluated using TEM images and dynamic light scattering (DLS) relative to hydrodynamic diameters. According to TEM analysis, the diameters for AuNPsp-PEG, AuNPst-PEG, and AuNPr-PEG were 48.20 ± 12.8 nm, 77.72 ± 16.05 nm and 36.10 ± 3.96 nm x 1.41 ± 1.48 nm (length x width), respectively. DLS analysis was used to determine the diameters of AuNPsp-PEG, AuNPst-PEG, and AuNPr-PEG, which were 146.73 ± 4.24 nm, 109.61 ± 1.27 nm, and 54.58 ± 0.34 nm × 8.47 ± 0.22 nm (length × width), respectively. The zeta potentials were −5.7 ± 7.6 mV, 33.1 ± 12.0 mV and 11.0 ± 18.9 mV, respectively, for AuNPsp-PEG, AuNPst-PEG, and AuNPr-PEG ( Figure 1J-L and Table 1). Table 1. Hydrodynamic diameters and zeta potential of AuNPsp-PEG, AuNPst-PEG and AuNPr-PEG.  Regarding the PDI of AuNPs, the AuNP st -PEG showed more monodispersity than AuNP sp -PEG and AuNP r -PEG, leading to more polydispersity (Table 1).

Contrast Effect of AuNPs-PEG Using CT
To study how the contrast effect of AuNPs were in CT, different concentrations of AuNPs (0.001 to 4 mM) were tested. It was found that AuNPs exhibited an attenuation factor tendency, especially for high concentrations (1 and 4 mM). Additionally, the attenuation factor tendency in CT contrast images was compared for different morphologies of AuNPs ( Figure 2) and revealed that AuNP r contributes to greater attenuation than AuNP sp and AuNP st . Regarding the PDI of AuNPs, the AuNPst-PEG showed more monodispersity than AuNPsp-PEG and AuNPr-PEG, leading to more polydispersity (Table 1).

Contrast Effect of AuNPs-PEG Using CT
To study how the contrast effect of AuNPs were in CT, different concentrations of AuNPs (0.001 to 4 mM) were tested. It was found that AuNPs exhibited an attenuation factor tendency, especially for high concentrations (1 and 4 mM). Additionally, the attenuation factor tendency in CT contrast images was compared for different morphologies of AuNPs ( Figure 2) and revealed that AuNPr contributes to greater attenuation than AuNPsp and AuNPst. For 4 mM, AuNPr showed greater X-ray attenuation than others, with CT values almost double those of the other AuNPs.

Cellular Uptake of AuNPs-PEG
Cells were treated with different treatments of AuNPs during 24 h. After that, cellular uptake of AuNPs linked with rhodamine were observed by the red fluorescence signals inside of cellular cytoplasm, as shown in Figure 3.
AuNPr, gold nanorods. For 4 mM, AuNPr showed greater X-ray attenuation than others, with CT value most double those of the other AuNPs.

Cellular Uptake of AuNPs-PEG
Cells were treated with different treatments of AuNPs during 24 h. After that, cell uptake of AuNPs linked with rhodamine were observed by the red fluorescence sig inside of cellular cytoplasm, as shown in Figure 3. AuNPs were found to be intracellular, indicating that AuNPs were internalized endocytosis. It was observed that AuNPs were stored on cytoplasm for 24 h, in both lines analyzed. However, more studies should be performed to understand uptake endocytosis.

AuNPs-PEG Decreased Cellular Viability
To evaluate the effect of AuNPs on cell viability, various cell lines were treated w different concentrations (0 to 1 mM) of AuNPs for 24 h to 72 h ( Figure 4). AuNPs were found to be intracellular, indicating that AuNPs were internalized by endocytosis. It was observed that AuNPs were stored on cytoplasm for 24 h, in both cell lines analyzed. However, more studies should be performed to understand uptake via endocytosis.

AuNPs-PEG Decreased Cellular Viability
To evaluate the effect of AuNPs on cell viability, various cell lines were treated with different concentrations (0 to 1 mM) of AuNPs for 24 h to 72 h ( Figure 4).
Cells were treated with different concentrations of AuNPs for 24 h. Then, the cells were exposed to a fraction of 2.5 Gy for three days, reaching a cumulative dose of 7.5 Gy of 6 MV photon beam. After 24 h of IR, the cell viability was measured, and the results are shown in Figure 5. We observed that most AuNPs were dose-dependent and did not increase the cytotoxic effects, except for 1 mM AuNP r -PEG. On day 1, AuNPs inhibited cellular viability with and without IR. On day 2, cells had more approximately 24 h to recover from the damages caused by the first IR. However, cells treated with AuNPs showed the capacity to repress the cell viability. On day 3, PC3 and DU145 cells demonstrated a tendency of reduced viability compared to the control group (0 Gy). Only DU145 cells treated with AuNP sp -PEG showed increased viability with 2.5 Gy. With respect to LNCaP cells, their viability decreased with AuNPs treatments compared to the control group in the first and second IR fractions. Comparing the three AuNP conformations used to treat the cells, AuNP r -PEG demonstrated viability reduction and dose dependence with repeated IR for three days. After this assay, the 0.1 mM concentration was selected to realize the next experiments.  , and then they were exposed to a cumulative dose of 7.5 Gy in three fractions of 6MV photon beam. An indication of cell viability was obtained by analyzing the overall metabolic activity of the cell population by PrestoBlue™ assay. Results were expressed as the mean ± SD, n = 6. AuNPsp-PEG, PEGylated spherical gold nanoparticles; AuNPst-PEG, PEGylated gold nanostars; AuNPr-PEG, PEGylated gold nanorods. The results were considered to be statistically significant when * p <0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The treatment groups were compared to the control group of the respective day, represented by black lines.
Cells were treated with different concentrations of AuNPs for 24 h. Then, the cells were exposed to a fraction of 2.5 Gy for three days, reaching a cumulative dose of 7.5 Gy of 6 MV photon beam. After 24 h of IR, the cell viability was measured, and the results are shown in Figure 5. We observed that most AuNPs were dose-dependent and did not increase the cytotoxic effects, except for 1 mM AuNPr-PEG. On day 1, AuNPs inhibited cellular viability with and without IR. On day 2, cells had more approximately 24 h to recover from the damages caused by the first IR. However, cells treated with AuNPs showed the The cells were previously treated with different concentrations of AuNPs (0-1 mM), and then they were exposed to a cumulative dose of 7.5 Gy in three fractions of 6MV photon beam. An indication of cell viability was obtained by analyzing the overall metabolic activity of the cell population by PrestoBlue™ assay. Results were expressed as the mean ± SD, n = 6. AuNP sp -PEG, PEGylated spherical gold nanoparticles; AuNP st -PEG, PEGylated gold nanostars; AuNP r -PEG, PEGylated gold nanorods. The results were considered to be statistically significant when * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The treatment groups were compared to the control group of the respective day, represented by black lines.
In an overview, the cells treated with the respective AuNPs until 0.1 mM maintained approximately 80% of cellular viability, supporting the negligible cytotoxicity towards the cells and suggesting their potential for RT therapeutic applications. and the final distance of the cell gap created on cell monolayers. Previously, the cells were treated with different conformations of AuNPs at 0.1 mM for 24 h. Then, the scratch of the cell culture was made. The gap closure was observed during 24 h, 48 h and 72 h for PC3, DU145, and LNCaP cells, respectively. After 24 h, the effect of the AuNPs on the gap size for each cell line was compared to the untreated control. As seen in Figure 5A-D, for the PC3 cell line, only AuNPr-PEG tended to delay the migration of cancer cells without receiving IR (~10%) and 24 h after the third fraction (3 × 2.5 Gy) by ~18%.  . The cells were treated with different concentrations (0-1 mM) for 24 h before being exposed to a cumulative dose of 7.5 Gy in three fractions of 6 MV photon beam. Relative migration (%) was measured 0, 6, 12 and 24 h after scratching. The results are expressed as the mean ± SD of 3 replicates. AuNP sp -PEG, PEGylated spherical gold nanoparticles; AuNP st -PEG, PEGylated gold nanostars; AuNP r -PEG, PEGylated gold nanorods.

Injury Assay with AuNPs-PEG
This assay evaluated the effect of AuNPs on cell migration by comparing the initial and the final distance of the cell gap created on cell monolayers. Previously, the cells were treated with different conformations of AuNPs at 0.1 mM for 24 h. Then, the scratch of the cell culture was made. The gap closure was observed during 24 h, 48 h and 72 h for PC3, DU145, and LNCaP cells, respectively. After 24 h, the effect of the AuNPs on the gap size for each cell line was compared to the untreated control. As seen in Figure 5A-D, for the PC3 cell line, only AuNP r -PEG tended to delay the migration of cancer cells without receiving IR (~10%) and 24 h after the third fraction (3 × 2.5 Gy) by~18%.
Regarding DU145 ( Figure 5E-H), without IR, only AuNP st -PEG showed a tendency to decrease the migration by around 8%. When cells were submitted at two or three fractions of IR, AuNP sp -PEG, AuNP st -PEG, and AuNP r -PEG tended to exhibit reductions of 15%, 4.6%, 18% for 2 × 2.5 Gy and 9%, 4% and 6% for 3 × 2.5 Gy, respectively. In LNCaP cells ( Figure 5I-L), AuNPs did not significantly influence migration, but without IR, AuNP st -PEG and AuNP r -PEG seemed to decrease migration by 4.7% and 4.4%, respectively. When cells were irradiated, the first fraction exhibited migration stimulation of~13-21%, but stimulus decreased slightly with the second fraction, by around 2.7-5.6%, and it was AuNP st -PEG that corresponded to the highest reduction in the stimulus (not significant). After the third fraction of IR, no differences were observed when comparing the IR groups to the control groups.

Sensibilization Effect of AuNPs-PEG in PCa Cells
After treatment with AuNPs for 24 h, radiosensitization was quantified using a clonogenicity assay. The results are shown in Figure 6.  In PC3 cells, treatment with either conformation of AuNPs tends to increase radiosensitization. However, in DU145, only cells treated with AuNP sp -PEG and AuNP st -PEG were shown to be slightly affected. For a cumulative 7.5 Gy dose, all AuNPs contributed to a slight decrease in the survival fraction. Treatment with AuNP r -PEG appeared to stimulate cell growth until cumulative 5 Gy. When LNCaP cells were treated with AuNP sp -PEG and AuNP r -PEG without IR, a reduction in the survival fractions was observed. After the first IR, LNCaP cells without AuNP treatment lost their capacity to grow into a colony. Regarding sensitivity enhancement ratio (SER) values (Table 2), for PC3 cells, all AuNP treatments caused an increase in cellular damage with IR, but AuNP st -PEG, AuNP r -PEG and AuNP sp -PEG produced a more significant increase in SER with doses of 2.5, 5, and 7.5 Gy (SER 2.09, 1.7 and 2.5, respectively), compared to the corresponding radiation group.
For DU145 cells, only AuNP r -PEG with 2.5 Gy exhibited a mild increase in sensibilization (SER 1.23) and all AuNPs showed an improved SER with 7.5 Gy (SER AuNP st -PEG-1.64; SER AuNP r -PEG-1.19 and SER AuNP sp -PEG-1.12) compared to the respective IR group. Moreover, LNCaP cells demonstrated a higher growth inhibition after 2.5 Gy with AuNP st -PEG (SER 3) and a smooth increase with AuNP sp -PEG (SER 1.2) compared to the 2.5 Gy radiation group.

Apoptosis Assay with AuNPs-PEG
The influence of AuNPs on cellular death with and without IR was evaluated using Annexin V-CF Blue/7-aminoactinomycin D (7-AAD)- Figure 7.
In PC3 cells, only AuNP r -PEG significatively reduced the cellular viability and increased the death cell percentage with IR (3.2%) and without IR (3.1%) compared to the respective control groups. When cells were treated with AuNPs, necrosis events were also observed. For AuNP sp -PEG and AuNP st -PEG, the cellular death values were similar to the control groups. Regarding DU145, the results showed a tendency for AuNP st -PEG to increase cellular death through apoptosis without IR. However, when cells were submitted to IR, the cellular death results were similar to the control group. Concerning the LNCaP cells, all AuNPs increased cellular death, mainly by necrosis without IR treatment, while with IR, the increase in cell death seems to be mainly induced by apoptosis.

ROS Assay with AuNPs-PEG
Overgeneration of ROS may cause DNA damage during mitochondrial respiration. ROS levels were detected by H2DCFDA to determine mitochondrial damage during exposure to AuNPs and IR. Figure 8 shows that AuNPs tended to increase ROS production in PC3 and LNCaP cells; however, the results were not statistically significant. cells, all AuNPs increased cellular death, mainly by necrosis without IR treatment, while with IR, the increase in cell death seems to be mainly induced by apoptosis.

ROS Assay with AuNPs-PEG
Overgeneration of ROS may cause DNA damage during mitochondrial respiration. ROS levels were detected by H2DCFDA to determine mitochondrial damage during exposure to AuNPs and IR. Figure 8 shows that AuNPs tended to increase ROS production in PC3 and LNCaP cells; however, the results were not statistically significant.  Otherwise, AuNP sp -PEG and AuNP r -PEG showed a tendency to decrease ROS production in DU145 cells. However, when PC3 cells were irradiated with 2.5 Gy, AuNP sp -PEG and AuNP r -PEG contribute to reduce ROS production significatively and AuNP st -PEG demonstrated a predisposition to increase the ROS production but was not significant. Additionally, AuNP sp -PEG and AuNP r -PEG exhibited a tendency to decrease ROS production in the DU145 cell line. Regarding LNCaP cells, AuNP sp -PEG, AuNP st -PEG, and AuNP r -PEG revealed a decreased of ROS production, but only AuNP sp -PEG and AuNP r -PEG had statistical significance.

Discussion
In the literature, only a few studies have explored the biological interaction between different conformations (shapes/sizes) of AuNPs and IR [17,25]. The most widely used PCa epithelial cell lines in RT studies are PC3, DU145 and LNCaP, being derived from PCa bone, brain, and lymph node metastases, respectively [26]. Regarding radiosensitivity, LNCaP appears to be the most radiosensitive, followed by DU145 and PC3 cell lines [27,28]]. Therefore, analyzing different types of cells could help to obtain results more representative of a wide range of PCa, because tumors are known to be heterogeneous, and tumor cells could have distinct features, such as aggressiveness and hormonal dependence. To the best of our knowledge, this is the first study comparing the effect of different conformations of PEGylated AuNPs on PC3, DU145, and LNCaP cells with cumulative doses of IR. Different sizes and shapes of AuNPs have been explored for their potential to enhance RT, but there is still no consensus.
The most commonly studied AuNPs in this context are AuNP sp, and Dou et al. suggested that AuNPs with a size of 3-50 nm are in the optimal size range for CT imaging and RT [22]. It has been reported that AuNPs of~13 nm can be used in clinical X-ray theranostic applications and have a better effect on tumor growth [29,30]. The radiosensitization depends on, in addition to size and shape, the number of internalized AuNPs [31]. Chithrani et al. demonstrated that 50 nm AuNPs have the best cellular uptake in terms of both weight and number [32]. Additionally, other shapes such as AuNP r , AuNP st , triangles, and cubes have also been studied due to their unique optical and physical properties [10,18,23]. Therefore, the optimal size and shape of AuNPs for RT potentiation are still under investigation and may depend on the specific application and mode of action.
Our results from CT imaging demonstrated that AuNPs' attenuation factor was directly proportional to AuNP concentration. Studies have proposed that AuNPs could be a good candidate as contrast for CT imaging [33,34]. Our results are in accordance with literature results studying a concentration of contrast from 0 to 1 mM [22,34,35]. Previously, studies have reported conflicting results regarding the influence of the size of AuNPs as a contrast agent, but no information was found regarding the shape of AuNPs [22,33,36].
On the basis of TEM images, the intracellular biodistribution of AuNPs tested in cell cytoplasm was demonstrated. Size, shape, surface chemistry, and surface modifications of AuNPs have an important role in biodistribution [10,17,37,38]. A recent study indicates that mitochondrial damage also presents a risk to long-term cancer cell growth [39].
Herein, the effect on cellular viability was evaluated after different treatments of AuNPs-PEG, in which the size, shape, and concentration of AuNPs were variable. Our results indicated lower cytotoxicity levels of AuNP treatments in the three cell lines, as shown in Figure 4, and in agreement with other studies [13,40,41]. The effect of AuNP conformation on cellular viability differs from PC3, DU145 and LNCaP cells, which could have different endocytosis capacity of AuNPs per unit volume. LNCaP cells have lower cellular volume and are less radioresistant than PC3 and D145 cell lines [20]. Therefore, the efficacy of cell endocytosis/uptake of AuNPs could influence the results. Of the concentrations tested, it was decided to use 0.1 mM for the following experiments: migration, survival curve, apoptosis, clonogenic and ROS assays [30,42].
Our results showed a tendency to reduce migration, which is in accordance with the literature [43][44][45][46]. Cell migration could be affected by various AuNP characteristics, such as size, shape, and surface chemistry. AuNPs could interfere with cellular signaling pathways that affect cell adhesion, and actin cytoskeleton dynamics, leading to senescence or cell death [47,48]. Further research is needed to fully understand the impact of AuNPs on cell migration and the underlying mechanisms involved.
The clonogenic assay is widely used to investigate cell damage induced by radiation [49,50]. Our results showed that AuNPs improved the radiosensitivity of PCa cells ( Figure 6 and Table 2).
Some articles have shown that AuNP sp inhibit colony forming ability in pancreatic cancer cells, but others have identified no long-term effects on colony formation in PC3, DU145 and MCF-7 cell lines [51,52]. Concerning IR, AuNP sp (~12 nm) decreased the number of colonies formed when irradiated with 2 Gy. At the same time, non-irradiated cells treated with different concentrations of AuNP sp had a similar result in the control (untreated cells) [53]. Similarly, Zhu et al. combined X-ray (1 to 8 Gy) with simple AuNP sp (~20 nm) or modified them with galactose-pegylated AuNP sp (GAL-(SH-PEG-NH 2 )-AuNP sp (~34 nm) and both structures inhibited the colony formation, but the GAL-PEG-AuNP sp showed better results, indicating that it could enhance the radiation sensitivity of HepG2 cells to X-ray [54]. Moreover, the interference of AuNPs on cell signaling pathways could lead to cellular damage and consequently contribute to reduced colony formation. However, more studies should be performed to identify how AuNPs affect colony formation [47].
It was verified that all treatments with different conformations of AuNPs at 0.1 mM induced more cell death through apoptosis or necrosis. AuNP r -PEG seems to increase apoptosis in all cell lines analyzed. In addition, these results are in agreement with the viability results obtained for these cell treatments. AuNPs may activate apoptotic pathways through a variety of mechanisms, including the release of cytotoxic molecules and the activation of specific signaling pathways [55][56][57]. In general, the effect of AuNPs on cell death is a complex mechanism, and it is not fully understood yet [57].
Regarding ROS production, cells without IR showed no significant differences between AuNPs-PEG tested. However, our results revealed that most AuNPs, especially AuNP sp -PEG and AuNP r -PEG, decreased the ROS production in PC3 and LNCaP cells.
In the literature, some studies support the notion that AuNPs could have antioxidant properties decreasing ROS production, while other studies have said that AuNPs could increase ROS production in the cell. One way that AuNPs may decrease ROS production is by inducing cells to employ many enzymatic and non-enzymatic antioxidants to neutralize the effect of ROS and bring back cell homeostasis [52]. Another hypothesis is that AuNPs may also scavenge ROS directly, effectively balancing them and reducing their levels in cells [53,54,58]. Overall, the available evidence suggests that AuNPs may be a promising strategy for reducing oxidative stress and mitigating the damaging effects of ROS depending on the conformation of AuNP used. On the other hand, human epidermal keratinocyte cells (HaCaT) treated with AuNP r -PEG (16.7 nm × 43.8 nm) produced significant ROS production when compared to mercaptopropane sulfonate (MPS)-AuNP sp (20 nm), leading to an upregulation of apoptosis-related genes (TNFSF10, ANXA5, CASP1, and EGR1) and proteins (caspase 1) [59]. Another study verified that Calu-3 epithelial cells treated with hexagonal-AuNPs generated more ROS and pro-apoptotic markers (Fas, caspase 3, and caspase 9) when cells were treated with triangular or spherical AuNPs [60]. Further research is needed to fully understand how AuNPs can induce cell death and optimize their use in various applications.
Among the published articles, it is difficult to make a meaningful comparison between studies because there are many variables related to nanoparticles and radiation parameters, cell types and experimental methodologies. Our study showed promising outcomes in using AuNPs as radiosensitizers, which is consistent with previous reports [18,20,30,55,[61][62][63][64]. Additionally, our irradiation methodology differs from other published articles, because our irradiation scheme was divided into three cumulative fractions of 2.5 Gy in order to be able to bring our study to actual clinical RT treatment and understand the authentic behavior of AuNPs during the standard treatment of PCa. Typically, in vitro studies perform single irradiation with different doses, with most studies not reporting on cumulative doses [18,19,30,32].

Synthesis of AuNPsp-PEG, AuNPst-PEG and AuNPr-PEG and Characterization
AuNP sp were prepared using the method of Turkevich and his co-workers, using a HAuCl 4 .4H 2 O solution reduced and stabilized by NaCt [65]. AuNP st were obtained according to the protocol reported by Tian et al. using a principle of growing a seed solution with AgNO 3 and L-ascorbic acid [66]. Similarly, AuNP r were produced using a seed growth solution based on the work of Scarabelli et al. After synthesis, PEGylation was achieved by adding SH-PEG-NH 2 to the AuNPs solution [67]. After 24 h stirring, the solution was washed twice at 7500 rpm for 30 min.
After synthesis, samples were analyzed using an Evolution 200 Series spectrophotometer UV-VIS spectrophotometer (ThermoFisher Scientific Inc., USA). The absorption values were used to determine the concentration of species in the solution. Additionally, samples were examined by transmission electron microscopy (TEM, JEOL JEM 1400 TEM at 120 kV, Tokyo, Japan) and scanning electron microscopy (SEM) using FEI Quanta 400FEG ESEM/EDAX PEGASUS X4M equipment to validate the synthesis and morphology of AuNPs. Furthermore, the nanoparticles' size distribution and zeta potential were analyzed by dynamic light scattering (DLS) and zetasizer (Nano ZS, Malvern Instruments Ltd., Malvern, UK), keeping the samples at 25 • C.

In Vitro Attenuation Measurement in CT
Pegylated AuNPs were diluted in PBS at different concentrations of Au or iodine (reference group) from 0 to 4 mM. Iomeprol is a nonionic, monomeric iodinated contrast medium used in clinical practice. Air and PBS were used as control [68]. Samples were placed in 0.5 mL Eppendorf tubes, and CT images were obtained using a clinical Light Speed VCT CT imaging system (GE Medical Systems, Milwaukee, WI, USA). CT scanning parameters were the same as clinical practice for the abdominal area: slice thickness, 1.0 mm; pitch, 0.8; tube voltage, 120 kV; tube current, 101 mA; field of view, 500 × 500; and gantry rotation time, 1 s. Subsequently, attenuation measurements were evaluated by loading the digital CT images in a standard display program, allowing us to quantify the Hounsfield units (HU) related to CT contrast values.

Cell Culture
The PC3, DU145, and LNCaP cell lines used in this study were kindly donated by Cancer Biology and Epigenetics Group-Research Center, Portuguese Oncology Institute of Porto, Portugal. PC3 and LNCaP cells were cultured and maintained in RPMI-1650 media (Biowest ® ) and DU145 cells are maintained in MEM media (Biowest ® ), supplemented with 10% of fetal bovine serum (FBS, Sigma) and 1% penicillin/streptomycin (Sigma). Both cell lines were cultured and grown to~80% confluence and were then sub-cultured and maintained at 37 • C with 5% CO2 in a humidified environment. All treatments were performed in serum-free conditions.

Cellular Uptake Experiments
Rhodamine (20 mM) was conjugated with AuNPs and incubated for 2 h. Subsequently, AuNPs were centrifugated and washed three times to remove the excess. Rhodamine B isothiocyanate is a red fluorescent dye (Ex 560 nm/ Em 580 nm) that can be applied as a marker for a cell s endocytic activity. Cells were treated with 0.1 mM Rhodamine-labeled AuNPs for 24 h. Then, the cells were observed via fluorescence microscopy (Carl Zeiss, Germany) with Zeiss Axio Imager Z1 software. The nuclei of the cells were stained with blue fluorescent 4 ,6 -diamino-2-fenil-indol (DAPI, blue) and cells without rhodaminetagged particles were used as the negative control for the studies.

Cell Viability
The viability of cells treated with AuNPs was assessed by PrestoBlue™ assay. This allowed an indirect estimation of cell viability, due to resazurin being converted to resofurin by mitochondrial activity [69].
Cells were seeded (1 × 10 5 cells/mL) in 96-well plates. After 24 h, cells were exposed to several concentrations of AuNPs ranging from 0 to 1 mM for another 24 h. Cells were exposed to 6 MV photon beam with a dose per fraction of 2.5 Gy, and the process was repeated for three days until cells had received a total cumulative dose of 7.5 Gy, except for the control group. After the treatment, 10% (v/v) PrestoBlue™ reagent was added per well and incubated for 1 h at 37 • C in a humidified incubator. The absorbance was measured using a microplate reader (Spectra Max Gemini XS) at 550 nm.

Cell Migration
Cell migration may be assessed in vitro by wound injury assay. After reaching confluence, cells were scraped from the culture dish using a pipette tip. Then, cells were incubated for 24 h with different treatments, and the damage recovery was visualized and photographed under an inverted microscope (Nikon) at 200× magnification. Wound closure was measured at 0 h, 6 h, 12, 24 h and 48 h using ImageJ software (U.S. National Institutes of Health) and calculated using Equation (1):

Colony Assay and Sensitization Enhancement Ratio (SER)
Clonogenic assay (colony formation) is an in vitro cell survival assay based on the ability of a single cell to form a colony which consists of at least 50 cells. Clonogenic assay was performed to compare the effect of AuNPs and RT on cell death. Thus, cells were seeded, treated for 24 h, and irradiated the following day with a single dose of 2.5 Gy for three consecutive days using a linear accelerator until it reached a cumulative dose of 7.5 Gy. Immediately after IR, 250-1500 cells were seeded in each well and incubated at 37 • C with 5% CO 2 for 7 days. After colony formation, cells were washed, fixed with 4% (v/v) paraformaldehyde, and stained with 5% crystal violet (v/v). Colonies with at least 50 cells were counted manually and surviving fractions (SF) at each dose were calculated using Equation (2) The experimental data of cell survival were fitted using the linear quadratic (LQ) model using Prism 8.0 (GraphPad Software, CA, USA). The parameters from the LQ formula given in Equation (3) were analyzed, where D is the physical dose expressed in Gy, and α and β are the model constants.
Additionally, the radiosensitization effect of AuNPs on RT was evaluated by calculation of sensitizer enhancement ratio (SER, Equation (4)). The SER was calculated as the quotient of the survival fraction without AuNPs treatment and of that in the presence of AuNPs. The greater the SER, the greater the destruction of PCa cells in the presence of AuNPs at the same radiation dose, leading to a reduction in the number of RT treatments with fewer side effects.

Apoptosis and Necrosis
Annexin V-CF Blue/7-aminoactinomycin D (7-AAD) allows the discrimination of early apoptotic cell populations from late apoptotic or necrotic cells through flow cytometry analysis. Annexin V/7-AAD (ab214663, Abcam) was used according to the manufacturer's protocol to detect and quantify death cell after cells received different treatments for 24 h. Viable cells were double negative, early apoptotic cells were positive to Annexin V and negative to 7-ADD, while late apoptotic cells were double positive and necrotic cells were negative to Annexin V and positive to 7-ADD. Cells were treated with three fractions of 2.5 Gy. Subsequently, cells were detached, washed twice with PBS, resuspended in 1x annexin-binding buffer, and incubated with 5 µL Annexin V-FITC and 5 µL 7-AAD for 15 min at 37 • C. Events were acquired with a FC500 cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA), and the acquired data were exported and analyzed with Flowjo software v10.4.2 (FlowJo LLC, Ashland, OR, USA). A total of 50,000 cells were evaluated.

Reactive Oxygen Species
The reactive oxygen species (ROS) was evaluated by the conversion of 2 ,7 -dichloroflu orescin diacetate (DCFDA/H2DCFDA) to 2 ,7 -dichlorofluorescein (DCF), a fluorescent compound (Kit ab113851, Abcam) by the presence of ROS. Briefly, 2.5 × 10 4 cells/well were cultured then washed with buffer and stained with DCFDA for 45 min at 37 • C in the dark. Next, cells were rewashed in buffer and treated with 0.1 mM of AuNPs-PEG. After 24 h, cells were immediately analyzed on a fluorescence plate reader at excitation/emission of 485/535 nm in endpoint mode in the presence of buffer.

Cell Irradiation/Irradiation Setup
To calculate the density of the 6-, 24-and 96-well plates, computed tomography scans were performed to obtain three-dimensional (3D) images. A water phantom with a thickness of 5 cm was placed on top and under the plates to simulate a biological structure and provide sufficient backscatter radiation to form an electric equilibrium ( Figure 9A). The dosimetric plan was performed using the software XIO-Release version 4.70.02 and was prescribed to the isocenter, using two fields (one anteroposterior and one posteroanterior, Figure 9B). The fractionation applied was 2.5 Gy in three days until a total radiation dose of 7.5 Gy was completed. Cells were irradiated with a 6 MV photon beam generated by PRIMUS linear accelerator (Siemens) in the radiotherapy department in Centro Hospitalar de São João (Porto, Portugal). The control group did not receive IR. The dosimetric plan was performed using the software XIO-Release version 4.70.02 and was prescribed to the isocenter, using two fields (one anteroposterior and one posteroanterior, Figure 9B). The fractionation applied was 2.5 Gy in three days until a total radiation dose of 7.5 Gy was completed. Cells were irradiated with a 6 MV photon beam generated by PRIMUS linear accelerator (Siemens) in the radiotherapy department in Centro Hospitalar de São João (Porto, Portugal). The control group did not receive IR.

Statistical Analysis
All experiments were performed in triplicate. The results are presented as mean ± standard deviation (SD). Data were analyzed with Prism 8.0 (GraphPad Software, CA). Differences between treatments were evaluated by Student's t-test or two-way ANOVA with Sidak multiple comparisons test, in accordance with the number of conditions and treatments. Results were considered significant when * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Conclusions
In conclusion, we demonstrated AuNPs to be potential IR enhancing agents and to show distinct responses depending on the cell lines. AuNP st -PEG and AuNP r -PEG decreased cellular viability in a dose-dependent manner. Our results showed a tendency of AuNP st -PEG and AuNP r -PEG to reduce migration and colony formation with and without IR. However, more studies in vitro are required to better understand the cellular mechanism responses and the AuNPs mechanism for radiosensitization. Additionally, AuNP studies with human blood samples should be performed to evaluate the thrombogenicity and hemocompatibility of AuNPs. Taking into account the results obtained in our study, in vivo studies should be performed in the future to evaluate the effects of AuNP treatments on prostate cancer animal models. Studies have reported that AuNPs can produce a wide variety of adverse reactions, impacting numerous organs, including skin, mucosa, kidney, blood, bone marrow, lungs, the nervous system, and the liver. Ultimately, if all trials are correctly evaluated and demonstrate promising results, human clinical research trials should also be considered from phases 1 to 3. In the future, AuNPs can be used in clinical practice, lessening side effects, while safety concerns should always be considered before clinical implementation.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
All data that support the findings of this study are available within the article or from the corresponding authors upon reasonable request.